Evidence that the receptor for C4a is distinct from the C3a receptor.
The cDNAs encoding the human (hC3aR) and mouse C3a receptors (mC3aR) were functionally expressed in RBL-2H3 cells. A calcium mobilization assay was utilized to assess the biologic activity of human anaphylatoxins, and C3a synthetic peptide agonists on hC3aR and mC3aR cells and this activity was compared to the activity of the anaphylatoxins on human neutrophils. Both hC3aR and mC3aR cells responded in a concentration-dependent manner with a robust calcium mobilization response to C3a with 50% effective concentrations (EC50s) of 0.24 nM and 1.3 nM, respectively. The response obtained with hC3aR cells was similar to the response elicited by C3a on human neutrophils (EC50 0.77 nM). The potency of a C3a analogue synthetic peptide (WWGKKYRASKLGLAR), derived from the fifteen carboxy-terminal residues (63-77) of C3a, relative to C3a, in stimulating calcium mobilization differed on cells expressing the human vs. mouse receptors. While the peptide was approximately 10 fold less active than C3a in stimulating calcium mobilization on cells expressing the hC3aR (EC50 2.0 nM), the peptide was essentially equipotent to the native ligand when tested on cells expressing the mC3aR. Data obtained with C4a, purified from activated serum, were difficult to interpret due to possible trace contamination of the C4a with C5a. Subsequently, an alternative C4a isolation scheme was utilized, via cleavage in vitro of purified C4. Concentrations of this latter C4a preparation, of up to 3.3 microM, had no effect on calcium mobilization in human neutrophils or in cells stably expressing the cloned C3a receptors, an indication that C4a does not interact with the C3a receptor.
Ames
R S
RS
Department of Molecular Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.
Tornetta
M A
MA
Foley
J J
JJ
Hugli
T E
TE
Sarau
H M
HM
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000032:analysis; D002460:Cell Line / Q000187:drug effects; D003001:Cloning, Molecular; D015926:Complement C3a / Q000494:pharmacology*; D015934:Complement C4a / Q000494:pharmacology*; D018076:DNA, Complementary / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D006801:Humans; D016177:Macrophage-1 Antigen; D051379:Mice; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000187:drug effects* / Q000276:immunology; D011951:Receptors, Complement / Q000235:genetics* / Q000276:immunology
D018076:DNA, Complementary; D016177:Macrophage-1 Antigen; D011951:Receptors, Complement; D015926:Complement C3a; D015934:Complement C4a; D002118:Calcium